Protocolo da aula:

Zebrafish  laboratory practical class

Objectives

·         Recognize and identify stages of development in a zebrafish embryo

·         Dechorionate embryos for experimental procedures

·         Learn how temperature affects zebrafish development

·         Identify and learn the importance of mutants and transgenic reporter lines

Materials:

·         Gloves

·         Petri dishes

·         Pipettes

·         Dissecting microscope

·         Zebrafish embryos

·         Zebrafish larvae (wt, mutants and transgenics)

·         Embryo medium

·         Tricaine

·         Needle

·         Swab

·         forceps

 

1-   Characterizing Developmental Stages of Zebrafish Embryos

Examining embryos:

1.1-     Using a pipette, place two or three embryos into a Petri dish with warm (28,5˚C) embryo medium.

1.2-     Examine the embryos in the Petri dish under a dissecting microscope. Use a pair of tweezers to gently roll the embryos over to observe them better.

1.3-     Use the figures to identify the stage of development of the embryos and estimate when they were fertilized.

1.4- Sketch the embryos and identify the structures that you observed.

 

Examining larvae:

1.1-     Using a pipette, place one or two larvae into tricaine solution to anesthetize the fish.

1.2-     Immediately after larva has stopped swimming, verify if it is responding to touch stimulus using a pair of tweezers.

1.3- Observe the larva under a dissecting scope, using a pair of tweezers to gently move the larva and sketch the larva, identifying the structures.

Questions

1- At what stage is the zebrafish embryo after 24 hours of development at 24ºC? 1.1- List the structures that you can see in the embryo at this point.

2 - What characteristics can you pick out? Somites? Eyespots? Pigment cells? Caudal fin? Notochord?

3- Does the zebrafish embryo respond to gentle touch stimuli? If it does, how so?

4 - Why do so many of the embryos die within the first few days postfertilization?

 

2 –Dechorionation of zebrafish embryos

2.1- Carefully grab the chorion of the embryo with a pair of fine-tip tweezers, without disrupting the embryo itself.

2.2- Grab the other end of the chorion with a second pair of fine-tip tweezers.

2.3- Gently pull the tweezers in opposing directions. Note: For blastula stage embryos, be very careful not to let the chorion touch the embryo, as this will disrupt the cells and kill the embryos.

2.4- Clean debris from bottom of petri dish.

2.5- Replace 3/4 of embryo medium in each petri dish.

Questions

1-On what day should the embryos be dechorionated?

 

3 - Observation of zebrafish mutants

3.1- Using a pipette, place one or two larvae into tricaine solution to anesthetize the fish.

3.2- Immediately after larvae have stopped swimming, verify if it is responding to touch stimulus using a pair of tweezers.

3.3- Observe the larvae under a fluorescence scope, using a pair of tweezers to gently move the larva and compare WT and mutants and identify the mutant phenotype.

Questions

1-What do you think could be the function of the normal alternative of the gene that is mutated in your mutant fish?

 

4 – Observation of transgenic larvae.

4.1- Using a pipette, place some larvae into tricaine solution to anesthetize the fish.

4.2- Immediately after larvae have stopped swimming, verify if it is responding to touch stimulus using a pair of tweezers.

4.3- Observe the larva under a fluorescence scope dissecting scope, using a pair of tweezers to gently move the larva and screen the transgenic larvae only.

4.4- Identify the transgenic line.

Questions

1-Why do we use transgenic reporter lines?